Vaginal Hydrogel

ABSTRACT

The vaginal hydrogel may be a composition which includes a glycosaminoglycan, a reactive molecule, and, in some embodiments, a therapeutic agent. The glycosaminoglycan and the reactive molecule may include either thiol groups or thiol reactive sites and have a pH that is within a range of a normal vaginal environment. Some of the thiol groups may interact with the vaginal mucosa and allow the hydrogel to remain in the vagina for a longer period than existing compounds intended for intravaginal administration. Therapeutic agents may be included in the composition. In these embodiments, the hydrogel acts as a vehicle which delivers the therapeutic agent.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to co-pending U.S. Provisional PatentApplication No. 62/512,588 filed on May 30, 2017 which is herebyincorporated by reference in its entirety.

BACKGROUND Field of the Invention

This disclosure relates to vehicles for drug delivery, and morespecifically, hydrogels.

Background of the Invention

Vaginitis affects millions of women around the world every year, andbacterial vaginosis is the most common form. Currently, marketedtreatments are either intravaginal creams and gels or systemic oraltreatments, and the most common drugs are metronidazole, clindamycin,and tinidazole. Metronidazole is currently the most common drug due tothe high efficacy of treating harmful bacteria, along with the lack ofeffect on beneficial lactobacilli. Some of the problems, however, withcurrent treatments include the messiness of intravaginal gels andcreams, the potentially lowered efficacy of the drugs in gels and creamsas the drug is lost through vaginal secretions, pH imbalances withproducts that do not have a pH of 3.5-5.0, and for systemic treatments,the problem of introducing the drugs to the entire body, which canproduce side effects.

Hydrogels have been utilized as a biomaterial in a variety of medicalapplications, owing to their high water content, similarity in physicalproperties to many tissues, and potential for incorporating drugs. Theseapplications include wound healing, tissue engineering and regenerativemedicine, drug delivery, and joint lubrication. These hydrogels can bebased on natural or synthetic materials, or a combination of the two.Typically, hydrogels will be crosslinked, either through physicalinteraction—such as simple entanglement, hydrogen bonding, or ionicinteraction—or through covalent bonds. Either method of forminghydrogels is typically done at or near what is considered standardphysiological pH (i.e., pH 7.4). However, many environments in the bodyare at a lower pH. In particular, the average pH of the vagina istypically between about 3.2 and 5.0, an environment that favorsmicroflora that are beneficial to the vagina. If the pH of the vagina isincreased, a shift in the balance of the microflora occurs, allowing forinfection and eventually leading to vaginosis or vaginitis.

Currently available treatments often treat dryness, an infection, orinflammation, but not multiple aspects simultaneously. Additionally,many of these treatments are provided in either a liquid or hydrogelformat, but not at a pH that is beneficial for the vaginal environment.Thus, there is a need for a hydrogel that can be delivered vaginallythat is at a pH targeted for this environment, namely between about 3.2and 5.0, and which may provide moisture as well as be used for deliveryof therapeutics.

BRIEF SUMMARY OF THE INVENTION

The hydrogels of the present disclosure are particularly useful intreating vaginal dryness, infection, and/or inflammation. Thesehydrogels may be provided in a composition that includes aglycosaminoglycan which has thiol groups and a molecule that has atleast 2 thiol-reactive sites.

The thiol groups and thiol-reactive sites may be in a molar ratio of atleast 2:1. The composition may have a pH between about 3.2 and about5.0. In some embodiments, the glycosaminoglycan is hyaluronic acid or amodified hyaluronic acid. In some embodiments, the molecule ispoly(ethylene glycol) diacrylate or poly(ethylene glycol)bisbromoacetate.

In some embodiments, the composition includes a glycosaminoglycan thathas thiol-reactive groups and a molecule that has at least 2 thiolgroups. In this embodiment, the thiol groups and thiol-reactive groupsmay be in a molar ratio of at least 2:1 and the composition may have apH between about 3.2 and about 5.0. In an example, the thiol-reactivesites may be acrylate, methacrylate, bromoacetate, iodoacetate,bromoacetamide, iodoacetamide, or maleimide. In an example, theglycosaminoglycan in this embodiment may be methacrylated or acrylatedhyaluronic acid. The molecule in this embodiment may be poly(ethyleneglycol) dithiol.

Some embodiments include a mucoadhesive agent. Examples of mucoadhesiveagents which may be included in the composition are as follows:methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, andhydroxypropylcellulose.

The disclosed compositions may be useful to deliver therapeutic agents,including antibacterial agents, antimicrobial agents, antiviral agents,estrogen, estrogen derivatives, and antiinflammatory agents.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following terms and phrases have the meanings indicated below,unless otherwise provided herein. This disclosure may employ other termsand phrases not expressly defined herein. Such other terms and phrasesshall have the meanings that they would possess within the context ofthis disclosure to those of ordinary skill in the art. In someinstances, a term or phrase may be defined in the singular or plural. Insuch instances, it is understood that any term in the singular mayinclude its plural counterpart and vice versa, unless expresslyindicated to the contrary.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,reference to “a substituent” encompasses a single substituent as well astwo or more substituents, and the like.

As used herein, “for example,” “for instance,” “such as,” or “including”are meant to introduce examples that further clarify more generalsubject matter. Unless otherwise expressly indicated, such examples areprovided only as an aid for understanding embodiments illustrated in thepresent disclosure, and are not meant to be limiting in any fashion. Nordo these phrases indicate any kind of preference for the disclosedembodiment.

While this invention is susceptible of embodiment in many differentforms, there are shown in the provided chemical structures, which willherein be described herein, several specific embodiments with theunderstanding that the present disclosure is to be considered as anexemplification of the principals of the invention and is not intendedto limit the invention to the illustrated embodiments.

The hydrogels described herein may be covalently crosslinked, or bothionically and covalently crosslinked. Covalent crosslinking may beachieved through such means as disulfide crosslinking, Michael-typeaddition, or photopolymerization. The hydrogel may be based on ananionic polysaccharide, which may be naturally anionic (such ashyaluronic acid or alginate) or may be modified to introduce or increasenegative charge, for example, through substitution of hydroxyl groupswith carboxyl groups. Particularly suitable are glycosaminoglycans, anda modified hyaluronic acid (hereinafter, “HA”). HA is a natural anionicpolysaccharide that is found throughout the body and has been shown tohave anti-inflammatory properties. Additionally, HA providesmoisturization and lubrication.

The hyaluronic acid may be modified to increase the number of negativecharges (e.g., carboxymethyl hyaluronic acid) for ionic crosslinking, orto include groups capable of covalent crosslinking. Examples ofmodifications include the addition of thiol or amine groups,thiol-reactive or amine-reactive groups, or photopolymerizable groups.These photopolymerizable groups may include methacrylate, acrylate, orvinyl groups. In the case of a thiol-modified HA, the hydrogel may beformed by disulfide crosslinking or by combining with a molecule havingthiol-reactive groups. In some embodiments, the hyaluronic acid has athiol modification of about 0.1 to about 1.0 μmol thiol/mg.

Thiol groups on the anionic polysaccharide are useful, as thiol groupsmay be beneficial for interacting with the vaginal mucosa, allowing thehydrogel to remain in the vagina for an extended period of time. Forexample, when the hydrogel composition has a thiolated polysaccharideand is combined with a thiol-reactive molecule, the ratio of thiolgroups to thiol-reactive groups will be about 2:1 or greater. This canallow for some thiols to be used for covalent crosslinking, while havingthiol groups remaining that may be used to interact with the vaginalmucosa.

The hydrogels may be formed at a pH between about 3.2 and 5.0, or may beformed at a higher pH, for example, between about 6.0 and 8.0, and thepH may be lowered once the hydrogel is formed. For forming hydrogels ata pH between about 3.5 and 5.0, particularly appropriate buffers includea lactic acid or citric acid buffer. A lactic acid buffer may beparticularly useful for the vaginal environment, as lactic acid/lactateis produced naturally in the vagina. Some types of crosslinking, forexample, via Michael-type addition, are typically done at a pH of about6.5-8.5, as the crosslinking proceeds extremely slowly outside of thisrange. However, to ensure proper hydrogel formation at lower pH, such asbetween 3.5 and 5.0, the crosslinking mixture may be placed in a sealedenvironment for several days until the hydrogel has formed.Additionally, the temperature of the environment may be increasedslightly during this time (to ˜35-40° C.) to facilitate crosslinkingwithout degrading the polysaccharide.

The resultant hydrogel may be loosely or tightly crosslinked by varyingthe degree of covalent and/or ionic crosslinking in the hydrogel. Thismay be accomplished by changing the concentration of the polysaccharide,an additional molecule that reacts with the polysaccharide to formcrosslinks, or the number of reactive groups on the polysaccharide oradditional molecule. The degree to which the hydrogel is loosely ortightly crosslinked may also be varied by changing the molecular weightof the additional molecule.

A therapeutic agent may be incorporated within the gel, and may beincorporated by mixing, covalently attaching the therapeutic agent to acomponent of the gel, or through ionic interaction with a component ofthe gel. For incorporation by mixing, the therapeutic agent may be mixedin either before or after the gel has formed. Therapeutic agents includeantimicrobials, including, but not limited to, antibiotics. In someembodiments, therapeutic agents may include antivirals. In otherembodiments, the therapeutic agents may include hormones, including butnot limited to, estrogen or an estrogen derivative. Still otherembodiments may include steroids and anti-inflammatories. Someembodiments may include combinations of therapeutic agents including,but not limited to, those listed as examples herein.

Other components may also be incorporated into the hydrogel to alterphysical properties of the resultant hydrogels, enhance mucoadhesion totissue, or both. Examples of mucoadhesive agents include cellulosederivatives, such as methylcellulose, hydroxymethylcellulose,hydroxyethylcellulose, and hydroxypropylcellulose. The cellulosederivatives may also lead to altered physical properties, such asenhanced viscosity, of the hydrogels.

The hydrogel may include a cationic polymer. In an example, the cationicpolymer may be polylysine.

In an embodiment, the composition includes a thiol-modified hyaluronicacid crosslinked to a poly(ethylene glycol) which has thiol-reactivesites. This composition may have a molar ratio of thiol groups tothiol-reactive sites between about 2:1 and about 3:1. The compositionmay have a pH between about 3.5 and about 4.5.

In an embodiment, the composition includes an anionic polysaccharidethat has thiol groups. The composition may further include a cationicpolymer and a molecule that has at least 2 thiol-reactive sites. Thethiol groups and thiol-reactive sites may be in a molar ratio of atleast 2:1. The composition may have a pH between about 3.2 and about5.0.

In another embodiment, the composition includes an anionicpolysaccharide that has thiol groups. The composition may include acationic polymer and at least a portion of the thiol groups may bedisulfide crosslinked. The composition may have a pH between about 3.2and about 5.0.

In another embodiment, the composition includes comprising an anionicpolysaccharide which has photopolymerizable groups. The composition mayfurther include a cationic polymer. In this embodiment, at least aportion of the photopolymerizable groups may be covalently crosslinked.The composition may have a pH between about 3.2 and about 5.0.

In another embodiment, the composition includes an anionicpolysaccharide that has thiol-reactive groups. The composition may alsoinclude a cationic polymer and a molecule that has at least 2 thiolgroups. In this embodiment, the thiol groups and thiol-reactive sitesmay be in a molar ratio of at least 2:1. The composition may have a pHbetween about 3.2 and about 5.0.

In another embodiment, the composition includes a glycosaminoglycanwhich has thiol-reactive groups and a molecule that has at least 2 thiolgroups. The thiol-reactive groups and thiol groups may be in a molarratio of at least 2:1 and the composition may have a pH between about3.2 and about 5.0.

A therapeutically effective amount of the composition according to theinstant disclosure may be provided in a kit for treating vaginosisand/or vaginitis. The kit is within the scope of the instant disclosure.

The instant disclosure also includes a method of treating or preventingvaginosis and/or vaginitis, including the step of administering atherapeutically effective amount of the composition disclosed herein toa subject.

EXAMPLES

The following examples are representative gels that have been formed.Thiol-modified carboxymethyl HA (CMHA-S) was synthesized as describedherein. The structure below is the chemical structure of thiol-modifiedcarboxymethyl hyaluronic acid (CMHA-S). For non-modified hyaluronicacid, R═H; for CMHA, R═CH₂CO₂H; for CMHA-S,R═CH₂—C(O)—NHNH—C(O)—CH₂CH₂SH.

Specifically, medical device-grade HA (900 kDa) was dissolved in 45%NaOH and stirred at room temperature for 2.5 hours. This mixture wasthen placed in isopropanol, and chloroacetic acid dissolved inisopropanol was added and allowed to react for 1 hour, then settle outof solution for 30 minutes. The liquid was decanted, and the resultantcarboxymethyl HA (CMHA) was dissolved in deionized (DI) H2O. The pH wasadjusted to 7.0, and the CMHA was purified using tangential flowfiltration (TFF).

3,3′-Dithiobis(propanoic dihydrazide) (DTP) was added to the purifiedCMHA solution and the pH adjusted to 4.75.N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDAC) was then added andthe pH maintained at 4.75 until a gel had formed, which was allowed toreact for a total of 4 hours. Dithiothreitol (DTT) was added, the pHadjusted to 8.5 and stirred overnight. The resultant thiolated CMHA(CMHA-S) was purified with TFF.

Poly(ethylene glycol)-bisbromoacetate (PEG-bba) was synthesized asdescribed herein. The structure below is the chemical structure ofpoly(ethylene glycol)-bisbromoacetate (PEG-bba).

Specifically, 1 mmol dry PEG (3350 Da) was dissolved in anhydrousdichloromethane. Triethylamine (2 mmol) was added, followed by dropwiseaddition of 4 mmol bromoacetyl chloride under argon. The reactionmixture was left to stir in the dark, under argon, overnight. Theproduct was precipitated in cold diethyl ether, filtered, and dried. ThePEG-bba was dissolved in deionized water and purified using dialysis(MWCO 1000), then lyophilized.

Methacrylated HA (MeHA) was synthesized as described herein. Thestructure below is the chemical structure of methacrylated hyaluronicacid (MeHA). Specifically, MeHA was synthesized by the dropwise additionof methacrylic anhydride (approximately 10-fold excess) to a solution of1% (w/v) HA (MW=350 kDa) in deionized water adjusted to a pH of 8-9 andreacted on ice for 24 h. The product was precipitated in cold 95%ethanol and filtered. The MeHA was dissolved in deionized water andpurified using dialysis, then lyophilized.

Example 1

A solution of CMHA-S (10 mg/ml) in deionized water was adjusted to a pHof 6.8. A solution of PEG-bba in distilled water (pH 6.8) was added toprovide a thiol-to-bromoacetate ratio of 2:1. The mixture was allowed toreact for 2 hours. The pH of the resultant loosely crosslinked gel wasadjusted to 3.5.

Example 2

A loosely crosslinked gel was prepared as in Example 1, except 1 ml of apolylysine solution (MW 150,000; 10 mg/ml) in deionized water was addedper 100 ml of CMHA-S solution and mixed for 30 minutes prior to addingthe PEG-bba solution. As in Example 1, this mixture was allowed to reactfor 2 hours, after which the pH was adjusted to 3.5.

Example 3

A solution of CMHA-S (10 mg/ml) in deionized water was adjusted to a pHof 3.8. A solution of PEG-bba in distilled water (pH 4.0) was added toprovide a thiol-to-bromoacetate ratio of 2:1. The mixture was allowed toreact for 3 days, at which time a loosely crosslinked gel had formedwith a pH of 3.9.

Example 4

A loosely crosslinked gel was prepared as in Example 3, except thatprior to adding the PEG-bba solution, methylcellulose (14 cPs) was mixedinto the CMHA-S solution to provide a final methylcelluloseconcentration of 5 mg/ml. The pH of the viscous, loosely crosslinked gelwas 4.0.

Example 5

MeHA is dissolved in deionized water (pH 4.5) to 15 mg/ml, andpolylysine (MW 150,000) is added to induce ionic crosslinking. After 30minutes of mixing, a photoinitiator (acetophenone in n-vinylpyrrolidone,3 mg/ml) is added and UV light (365 nm, 10 mW/cm²) used tophotocrosslink and form a gel.

Example 6

A solution of MeHA (15 mg/ml) in deionized water is adjusted to a pH of6.8. A solution of PEG-dithiol in distilled water (pH 6.8) is added toprovide a thiol-to-methacryl ratio of 2:1. The mixture is allowed toreact until a gel is formed. The pH of the gel is then adjusted to about4.0.

While specific embodiments have been illustrated and described above, itis to be understood that the disclosure provided is not limited to theprecise configuration, steps, and components disclosed. Variousmodifications, changes, and variations apparent to those of skill in theart may be made in the arrangement, operation, and details of themethods and systems disclosed, with the aid of the present disclosure.

Without further elaboration, it is believed that one skilled in the artcan use the preceding description to utilize the present disclosure toits fullest extent. The examples and embodiments disclosed herein are tobe construed as merely illustrative and exemplary and not a limitationof the scope of the present disclosure in any way. It will be apparentto those having skill in the art that changes may be made to the detailsof the above-described embodiments without departing from the underlyingprinciples of the disclosure herein.

I claim:
 1. A composition comprising at least one glycosaminoglycan,each of the at least one glycosaminoglycan comprising a plurality ofthiol groups; and a molecule comprising at least two thiol-reactivesites, wherein the plurality of thiol groups and at least twothiol-reactive sites are in a molar ratio of at least 2:1, and whereinthe composition has a pH between about 3.2 and about 5.0.
 2. Thecomposition of claim 1, wherein the at least one glycosaminoglycancomprises a modified hyaluronic acid.
 3. The composition of claim 2,wherein the modified hyaluronic acid comprises a thiol modification ofabout 0.1 to about 1.0 μmol thiol/mg.
 4. The composition of claim 3,wherein the modified hyaluronic acid comprises a thiol-modifiedhyaluronic acid; wherein the thiol-modified hyaluronic acid iscrosslinked to a poly(ethylene glycol), the poly(ethylene glycol)comprising a plurality of thiol-reactive sites; and wherein thecomposition comprises a molar ratio of thiol groups to thiol-reactivesites of between about 2:1 and about 3:1; and a pH between about 3.5 andabout 4.5.
 5. The composition of claim 1, wherein the at least twothiol-reactive sites are independently selected from the groupcomprising acrylate, methacrylate, bromoacetate, iodoacetate,bromoacetamide, iodoacetamide, and maleimide.
 6. The composition ofclaim 1, wherein the molecule comprises poly(ethylene glycol) diacrylateor poly(ethylene glycol) bisbromoacetate.
 7. The composition of claim 1,further comprising a cationic polymer.
 8. The composition of claim 7,wherein the cationic polymer comprises a polylysine.
 9. The compositionof claim 1, further comprising a mucoadhesive agent.
 10. The compositionof claim 9, wherein the mucoadhesive agent is selected from the groupcomprising methylcellulose, hydroxymethylcellulose,hydroxyethylcellulose, and hydroxypropylcellulose.
 11. The compositionof claim 1, additionally comprising a therapeutic agent.
 12. Thecomposition of claim 11, wherein the therapeutic agent comprises one ormore of the agents in the following list: an antimicrobial agent, anantibacterial agent, an antiviral agent, an anti-inflammatory agent, asteroid, an estrogen, and an estrogen derivative.
 13. A compositioncomprising: an anionic polysaccharide comprising a plurality of thiolgroups; a cationic polymer; and a molecule comprising at least twothiol-reactive sites; wherein the thiol groups and the thiol-reactivesites are in a molar ratio of at least 2:1; and wherein the compositionhas a pH of between about 3.2 and about 5.0.
 14. The composition ofclaim 13, wherein at least a portion of the thiol groups are disulfidecrosslinked.
 15. The composition of claim 13, wherein the anionicpolysaccharide comprises a modified hyaluronic acid.
 16. The compositionof claim 13, wherein the anionic polysaccharide comprises a modifiedglycosaminoglycan.
 17. The composition of claim 13, further comprising atherapeutic agent.
 18. A composition comprising: a glycosaminoglycancomprising a plurality of thiol-reactive sites; and a moleculecomprising at least two thiol groups; wherein the thiol reactive sitesand thiol groups are in a molar ratio of at least 2:1; and wherein thecomposition has a pH of between about 3.2 and about 5.0.
 19. Thecomposition of claim 18, wherein the glycosaminoglycan is methacrylatedor acrylated hyaluronic acid.
 20. The composition of claim 18, whereinthe molecule comprising at least two thiol groups consists ofpoly(ethylene glycol) dithiol.
 21. The composition of claim 18, furthercomprising a therapeutic agent.
 22. The composition of claim 18, furthercomprising a mucoadhesive agent.